MCT4 Expression Is a Potential Therapeutic Target in Colorectal Cancer with Peritoneal Carcinomatosis

Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.Department of Internal Medicine, Chungbuk National University Hospital, Chungbuk National University College of Medicine, Cheongju, Korea.

Introduction

Colorectal cancer represents the third most common cancer in males and the second in females worldwide, with 1.2 million new diagnoses and 608,700 deaths estimated in 2008 (1). The survival of metastatic colorectal cancer has been markedly improved in the past decades, resulting from the development of targeted therapies, such as cetuximab, panitumumab, bevacizumab, and regorafenib (2–6). However, tumor heterogeneity and selective pressure in patients with colorectal cancer leads to the development of resistance to anticancer treatments, and several studies have attempted to seek new therapeutic targets by addressing the colorectal cancer molecular landscape (7–10). In particular, the targeting of metabolic enzymes or transporters represents a different approach for cancer therapy and is expected to overcome the resistance to current therapies (11).

Monocarboxylate transporters (MCT) are proteins that are expressed in the cell membrane and control the lactate metabolic pathway. The level of lactate is increased in rapidly growing cancer cells (12, 13) as it is generated by glycolytic metabolism, on which the survival of cancer cell is largely dependent (14, 15). MCTs prevent intracellular acidification consequent to the increased intracellular lactate production in cancer cells by balancing its influx and efflux to maintain a steady intracellular pH and protect against loss of cancer cell viability (16). Particularly, MCT1 and MCT4 require the formation of complex with chaperone, CD147. CD147 is a transmembrane protein of the immunoglobulin superfamily and is also known as basigin (17–19).

The MCT family comprises 14 members in total, among which MCTs 1–4 play various roles in mammals (20). MCT1 and MCT2 take part in lactate uptake, thus initiating the oxidative pathway of lactate, whereas MCT4 functions in lactate excretion (21–23). Furthermore, the pattern of MCT expression differs according to cell type. MCT1, but not MCT2, expression has been shown in almost all tissues (23). Similarly, MCT expression in tumor cells depends upon tumor type; for example, MCT4 expression in lung cancer may be downregulated, whereas both MCT1 and 4 are generally upregulated in breast cancer (24). In addition, tumor microenvironment, such as high oxidative stress, lactate metabolism is the main determinant in controlling MCT expression (25, 26).

MCTs also correlate with cancer progression, infiltration, and angiogenesis, with cells exhibiting high MCT expression demonstrating invasiveness and poor prognosis in several solid tumors, including colorectal cancer (27). Accordingly, several studies have evaluated MCT inhibitors as anticancer treatments. For example, the MCT1/2 inhibitor AR-C155858 and MCT1 inhibitor AZD3965 showed preclinical anticancer activity (28, 29) and a phase I trial of AZD3965 for refractory and advanced solid tumors or lymphoma is ongoing (NCT07197595).

We previously studied the clinical implication of MCT4 in metastatic gastric cancer with peritoneal carcinomatosis, which revealed that the inhibition of MCT4 reduced tumor cell proliferation and the export of lactate (30). However, little information is available regarding the relationship between MCT expression and clinical outcome in colorectal cancer. In the current study, we investigated the expression patterns of MCTs in colorectal cancer cell lines, and their therapeutic potential as target proteins in the treatment of colorectal cancer. We also analyzed survival outcomes according to MCT4 expression in primary colorectal cancer tissues derived from patients to determine its prognostic impact on relapse-free and overall survival (OS).

To establish patient-derived cells (PDC) from patients with metastatic colorectal cancer and malignant effusion, the individuals who were enrolled onto the SMC Oncology Biomarker study (NCT#01831609) were screened for MCT expression by Western blot analysis. All patients provided an informed consent form according to the SMC Institutional Review Board (IRB) guidelines. Briefly, collected effusions (1–5 L) were divided into 50 mL tubes, centrifuged at 1,500 rpm for 10 minutes, and washed twice with PBS.

All cells and PDCs were grown in RPMI1640 medium supplemented with 10% FBS, an antibiotic, and an antimycotic (Invitrogen Corporation). Cells were cultured at 37°C in a humidified 5% CO2 environment.

All cell lines were tested for mycoplasma contamination and authenticated by STR DNA profiling (Samsung Medical Center Basic Research Support Center, Seoul, Korea) every 6 months, and all cells were cultured according to the manufacturer's manual and had been passaged for fewer than 2 months after thawing.

Real-time PCR was performed using a Prism 7900HT Sequence Detection System (PE Applied Biosystems). MCT1, MCT2, MCT4 mRNA and 18S rRNA were detected using TaqMan Gene Expression Master Mix Reagent and TaqMan probes (Applied Biosystems). Data were normalized using 18S rRNA as an endogenous control and calculated using the comparative Ct method (2ΔΔCt).

Cell growth assessment and colony formation assay

To assess cell numbers, cells (1 × 105 cells/6-well plate, Corning Costar Corp.) were transfected with siRNAs and incubated for 3 days. Adhered cells were trypsinized, stained with 0.2% trypan blue (Sigma), and counted using a hemocytometer. Cell proliferation following each treatment was compared with that of untreated cells.

For the clonogenic assay, cells were transfected with siRNAs for 24 hours, irradiated with a 137Cs source (2.01 Gy/minute, IBL-437C, CIS-US Inc.), trypsinized, and counted. Then, 400 cells were replated in 6-well plate. After incubation at 37°C for 10 to 14 days, colonies were stained with 0.005% (w/v) crystal violet (Sigma). Colonies (>50 cells) were counted and surviving fractions following the given treatments were calculated on the basis of the survival of nonirradiated cells.

ELISA assay

Secreted protein levels of VEGF and Angiopoietin 2 were assessed using culture media (200 μL) collected from siHIF1a or siMCT4-transfected cells. Protein levels of VEGF and Angiopoietin 2 were measured using an ELISA Kit for human VEGF and Angiopoietin 2 (R&D Systems), according to the manufacturer's instructions and quantified using a microtiter plate reader at 450 nm wavelength.

Xenograft study

Male BALB/c nude mice, 4 to 6 weeks old, were obtained from Orient Bio Inc. Mice were implanted subcutaneously with SW602 (5 × 106) cells in a 100 μL volume. The mice were randomized and the treatment started when the tumor size reached 60 mm3 at 11 days after inoculation. Mice were assigned into five groups: siC, siMCT1, siMCT2, siMCT4, or a combination of siMCT1 and siMCT4. siMCTs (1 μg with HiPerfect transfection reagent/tumor, intratumoral injection) were administered twice per week.

Tumor growth was measured using a digital caliper (Proinsa) every 3 to 4 days, and average tumor volumes were calculated using the following formula: V = (L × W2)/2, where V = volume (in cubic millimeters), L = length (in millimeters), and W = width (in millimeters). The mice were sacrificed and the tumors (three tumors per treatment group) were resected and frozen in liquid nitrogen until later use for Western blot analyses. All mouse experiments were conducted in accordance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals, and the protocols were approved by the appropriate IRBs at Samsung Medical Center (agreement- 20141211001).

Human colorectal cancer and PDCs

We collected 39 matched pairs of primary colorectal cancer and normal colorectal tissues and PDCs (n = 11) at the Samsung Medical Center. All colorectal cancer tumors and control tissues were confirmed by the hospital's Clinical Pathology Department. The normal colorectal tissue was collected at a distance of at least of 10 cm from the tumor site. To establish PDCs from patients with metastatic colorectal cancer and malignant effusion, individuals who were enrolled in the SMC Oncology Biomarker study (NCT#01831609) were screened for the expression of MCTs by Western blot analysis and real-time PCR.

All patients provided written informed consent. This study was performed in accordance with the Declaration of Helsinki and was approved by the IRB of Samsung Medical Center.

Patients for IHC and survival analysis

We collected data from the electronic medical records of patients who were diagnosed with colorectal cancer and who underwent curative surgery at the Samsung Medical Center between June 2008 and May 2009 (N = 586). Data including sex, node-metastasis, staging, location of tumor, histology, lymphatic invasion, perineural invasion, and vascular invasion were collected. This study was approved by the IRB of the Samsung Medical Center.

MCT4 IHC

IHC analysis was performed on tissue microarray blocks. Two tissue cores with a diameter of 2.0 mm from representative tumor areas marked by pathologists were punched from tumor tissue blocks and included in each array block. Formalin-fixed, paraffin-embedded blocks were cut into 4-μm serial sections. Antigen retrieval was performed for 20 minutes with pH 6.0 LOW buffer [EnVision FLEX Target Retrieval Solution Low pH (50×; Dako)] in a 97°C water bath. Endogenous peroxidase blocking was conducted for 5 minutes. The sections were incubated using a primary antibody to MCT4. MCT4 expression was evaluated by two independent pathologists (K.M. Kim and H. Bang). To determine MCT4 expression, the intensity and proportion of staining in the tumor cell membrane were assessed semiquantitatively. The intensity was classified into negative, weak, moderate, and positive and scored as 0, 1, 2, and 3, respectively. The proportion of stained tumor cells was scored on a scale 0 to 4 (0, <5%; 1, 5%–25%; 2, 26%–50%; 3, 51%–75%; 4, >75%).

Statistical analysis

Preclinical data were evaluated by a two-tailed t test or one-way ANOVA using GraphPad Prism version 4.01. For analysis of the MCT4 IHC cohort, SPSS statistical software version 23 was used. All comparisons were examined by the χ2 test or Fisher exact test. Recurrence-free survival (RFS) was measured from the date of curative surgery to the date of first cancer recurrence or death. OS was measured from the date of curative surgery to the date of death or date the patient was last seen. The Kaplan–Meier method was used to estimate RFS and OS, and survival curves were compared by the log-rank test. To evaluate the prognostic value of MCT4, we utilized a multivariable Cox proportional hazards regression model. All tests were two-tailed and P values <0.05 were considered statistically significant.

The proliferation of colorectal cancer cell lines decreased following MCT inhibition by transfection with siRNA (Fig. 1B; Supplementary Fig. S1). MCTs were effectively knocked down by siMCT1, 2, or 4 after 72 hours, and cell proliferation was significantly decreased depending on the type of MCTs expressed in each respective cell line. In particular, MCT1 expression was confirmed by Western blot analysis in 5 cell lines (CoLo320, NCI-H716, SW620, SW480, and DiFi), which showed decreased proliferation by the addition of siMCT1. MCT2 was expressed in NCI-H716 and SW480 cell lines, and transfection with siMCT2 hindered the expression of MCT2 as well as cell proliferation in these lines. Similarly, SW620 and SW480 cell lines exhibited MCT4 expression, and their cell proliferation was inhibited by siMCT4. Figure 1C illustrated that normal colorectal cell line (CCD-18Co) did not express MCT4 and was not affected by siMCT4. These results suggested that knockdown of MCT1, 2, and 4 inhibited cancer cell proliferation, which expressed MCT1, 2, and 4, respectively.

Effect of MCT inhibition on colorectal cancer growth in vivo

To determine whether the antitumor effect of MCT inhibition could also be observed in vivo, we implanted SW620 cells, which expressed MCT1 and 4, into mice and assigned them to the following 5 groups (n = 5 mice/treatment group): untreated control, siMCT1, siMCT2, siMCT4, and the combination siMCT1 and 4. Mice were subjected to two subcutaneous injections in each flank to promote the growth of two tumors (10 tumors/treatment group). The combination siMCT1 and siMCT4 treatment resulted in the most significant decrease of tumor volume on day 17 [siControl vs. siMCT4, mean tumor volume on day 17, 857.5 vs. 381.2 cm3; mean difference, 476.3 cm3; 95% confidence interval (CI), 320.4–632.3; P < 0.0001, Fig. 2A]. A single treatment with siMCT1 or siMCT4 but not siMCT2 also showed a significant reduction of tumor volume.

Effect of MCT knockdown on tumor growth in vivo. BALB/c nude mice were injected subcutaneously in the bilateral flank with SW620 cells (5 × 106 cells). One week after injection, mice were treated 2 times per week with an intratumoral injection of siMCTs (1 μg with HiPerfect transfection reagents/tumor). A, Time course of tumor growth following injection of siMCTs (left) and mean tumor volume and SD (right). B, Cell proliferation in xenograft of SW620 was analyzed by IHC with anti-PCNA antibody (Santa Cruz Biotechnology, PC-10, 1:2,000). The brown staining in the nucleus is the PCNA signal, and cell proliferation was decreased by siMCT1 and/or siMCT4. C, The levels of MCT proteins and basigin were decreased by siMCTs in Western blot analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

The xenograft tumors were resected, and protein expression was evaluated using IHC. IHC was performed by applying a monoclonal anti-PCNA antibody for the validation of tumor growth inhibition with SW620 in xenografts (Fig. 2B). The expression level of PCNA in IHC was lowest in the combination siMCT1 and MCT4 group; siMCT1 or siMCT4 alone also lessened the level of protein expression in the xenograft tumors. Similar results of protein expression were observed by Western blot analysis (Fig. 2C).

We next assessed the additive effects of MCT inhibition and radiotherapy or cytotoxic chemotherapy. SW620 cells were transfected with MCT siRNA and then irradiated with a 137Cs source or treated with 5-fluorouracil (5-FU) after 24 hours. As the dose of radiation increased, the cell proliferation became further decreased, demonstrating a radiation dose-dependent inhibition in cells with concomitant knockdown of MCT1 or 4 (Fig. 3A). Knockdown of MCT1 or 4 significantly induced cell death upon treatment of 5-FU, whereas in the case of MCT2, the level of cell proliferation was not significantly different than that of the control (Fig. 3B). The other tested cell lines (CoLo320, NCI-H716, and SW480) also demonstrated best additive effects of MCT inhibition with irradiation or 5-FU (Supplementary Fig. S2).

Additive effect of MCT inhibition with radiotherapy or chemotherapy. SW620 cells were transfected with indicating siMCTs; then, the cells were irradiated with a 137Cs source or 5-FU after 24 hours. A, Downregulation of MCTs is associated with the radiosensitizing effects. Radiosensitivity assessed by clonogenic cell survival. SW620 cell lines treated with siMCTs and radiation were plated (300 cells/well in 6-well plate) and maintained for 14 days. Colonies were stained with 0.1% crystal violet, and colonies larger than 50 cells were counted. Surviving fractions following the given treatments were calculated on the basis of the survival of nonirradiated cells. B, Additive effect of MCT inhibition with 5-FU. SW620 cell lines treated with siMCTs and 5-FU for 3 days and stained with 0.4% trypan blue. Results of cell counts are expressed as percentage of cell proliferation using the control as reference. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

We next investigated the MCT expression levels in 39 matched paired normal colon tissue, colorectal cancer tissue, and 11 PDCs acquired from malignant ascites using qRT-PCR, which demonstrated markedly increased MCT4 expression in PDCs from malignant ascites (Fig. 4A). IHC performed to evaluate the MCT4 expression in tissue samples and PDCs also showed strong expression in PDCs from malignant ascites (Fig. 4B). Treatment of 3 PDCs with siMCT4 indicated that knockdown of MCT4 also induced the reduction of MCT4 expression and cell death (Fig. 4C), similar to the effects in the colorectal cancer cell lines.

Correlation between MCT4 expression and hypoxia- or angiogenesis-related factors. A, Strong expression of MCT4 and hypoxia/angiogenesis-related factors in PDCs compared with normal and tumor tissue. B, Left, protein expression when siRNA of HIF1α and MCT4 were transfected on SW48 cell line, in hypoxic conditions with 1% of oxygen. The MCT4 expression was decreased by knockdown of HIF1α, but silencing of MCT4 could not reduce the HIF1α expression. Right, the transfection with vector of HIF1α induced the MCT4 expression. C, Angiogenesis-related factors, including VEGF and angiopoietin 2, were also decreased by siHIF1α or siMCT4 in PDCs with ELISA assay. Left two graphs show that VEGF and ANGPT2 were significantly decreased by siRNA of HIF1α and MCT4. Right two graphs show that the transfection with vector of HIF1α increased VEGF and ANGPT2. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

To determine the order of contribution of these factors to the underlying mechanism, we induced a hypoxic condition using 1% oxygen and transfected the SW48 cells with siHIFα or siMCT4. Hypoxia induced the expression of HIFα and MCT4, ANGPT2, and BiP expression were decreased after knockdown of HIFα. However, knockdown of MCT4 did not influence the expression of HIFα, and MCT4 expression was upregulated following overexpression of HIF1α (Fig. 5B).

We also measured the expression of the angiogenesis-related mediators, ANGPT2 and VEGF by ELISA in PDCs. Transfection with siHIFα or siMCT4 in the hypoxic condition in PDCs induced a decrease of VEGF and ANGPT2 expression (Fig. 5C). A similar phenomenon was also observed in other cell lines (HT29, HCT8) treated with siHIFα or siMCT4 (Supplementary Figs. S3 and S4).

Strong MCT4 expression by IHC is associated with significantly shorter RFS in patients with colorectal cancer

A total of 586 patients who had been diagnosed with colorectal cancer received curative surgery between June 2008 and May 2009. We compared baseline characteristics and survival outcome by dividing the patients into two groups, MCT4 positive and negative (Table 1). The MCT4-positive group was defined as exhibiting strong positive MCT4 expression by IHC (intensity score 3), and the MCT4-negative group had an intensity score of 0 to 2 by IHC (Fig. 4B). During the 83.5 months of median follow-up duration, the patients with positive MCT4 expression by IHC showed significantly shorter RFS than the MCT4-negative group (5-year RFS rate, 83.5% vs. 73.1%, P = 0.013, Fig. 6). Notably, in the multivariate analysis, the MCT4 positive state was identified as an independent risk factor for RFS (HR = 1.29; 95% CI, 1.11–1.49; P = 0.001).

Discussion

MCT expression has been known to correlate with tumor invasion or metastasis in several cancers in addition to colorectal cancer (31), including gastric (32), lung (33), breast (34), and prostate (35) cancers. In the current study, we showed that MCT proteins are expressed in colorectal cancer cell lines and tumor tissue and present the first demonstration that MCT4 expression was significantly elevated in malignant cells in colorectal cancer ascites. In our previous report regarding MCT4 in gastric cancer, MCT4 also was found to be overexpressed in malignant cells in ascites. However, MCT4 expression in gastric cancer was not revealed as a factor for poor prognosis in survival analysis (30). An association between MCT4 expression and poor survival has been reported in pancreas cancer (36) and clear renal cell carcinoma (37); in comparison, here we provide the first report that in patients with colorectal cancer, MCT4 expression instead represents a risk factor for early relapse and shorter RFS.

MCT4 is upregulated by hypoxia as a transporter and is known to be under the control of HIF1α (37, 38), which performs essential roles in pH regulation, metabolism, and cell invasion in addition to controlling VEGF and ANGPT2 in the hypoxia-signaling pathway (39). We identified significantly higher expression of HIF1α and MCT1, 2, 4, VEGF, and ANGPT2 in PDCs from malignant ascites in comparison with normal and tumor tissue. These results implied that hypoxic conditions in the peritoneal space induced metabolic adaptation, including upregulation of MCT4 in colorectal cancer with peritoneal seeding. We also verified HIF1α as an additional key upstream regulatory factor of MCT4 because HIF1α knockdown or upregulation led to reduced or increased expression of MCT4, respectively.

MCT4 also has been reported to be associated with VEGF in colorectal cancer (40). VEGF is usually upregulated and induced by angiogenesis and hypoxia in cancer cells (41) and is known as a therapeutic target of metastatic colorectal cancer for the anti-VEGF antibody, bevacizumab. Bevacizumab function depends on the presence of a specific genomic alteration and does not induce marked survival benefits. Recent studies reported the mechanisms between adaptive resistance to antiangiogenic treatments and metabolic symbiosis. Metabolic symbiosis based on the exchange of lactate and depends on MCTs is also responsible for tumor resistance to antiangiogenic treatments (42–44). In the current study, MCT4 inhibition was also able to downregulate VEGF expression in colorectal cancer cell lines. Therefore, an MCT4 inhibitor might have a potential role in the treatment of colorectal cancer along with bevacizumab.

We used RPMI1640 medium supplemented with 10% FBS for culture of all cells and PDCs. In the current study, not only MCT4 but also MCT1 and MCT2 inhibition demonstrated the suppression of tumor growth in the absence of exogenous lactate. However, lactate could be generated from metabolism of cancer cells and transported through the membrane. In case of MCT2, we previously reported that knockdown of MCT2 could suppress tumor growth in colorectal cancer in the absence of exogenous lactate (45).

According to the findings following clinical applications of MCT inhibitor (17, 29, 46), therapeutic approaches for targeting MCTs appear to hold promise. Here, we demonstrated that MCT knockdown was able to inhibit tumor cell proliferation and enhance the efficacy of radiotherapy or cytotoxic chemotherapy, which is consistent with the previous findings that hypoxia and lactate concentration are linked with the treatment failure of radiotherapy or chemotherapy in many cancers (47–49). Similarly, Sonveaux and colleagues demonstrated that MCT inhibition increased tumor radiosensitivity (50), and Bola and colleagues showed that the MCT1 inhibitor AZD3965 augmented radiosensitivity in the treatment of small-cell lung cancer (51).

Notably, our finding that elevated MCT4 expression in colorectal cancer as measured by IHC could serve as a negative prognostic factor reflected the observation that RFS was significantly worse with high MCT4 expression; however, no difference was noted in terms of OS (Supplementary Fig. S5). The association between MCT4 expression and shorter OS has previously been described in several studies involving colorectal cancer and other cancers (49, 52). One potential explanation for this discrepancy may be selection bias. The patients assessed in the current study were not diagnosed with metastatic disease, and all had received curative resection at the time of tissue archiving. As MCT4 expression is associated with metastatic and advanced disease, the survival analysis of MCT4 expression might be more conclusive in patients with metastatic colorectal cancer.

Metastatic colorectal cancer with peritoneal carcinomatosis carries a grave prognosis with a median survival of 6 to 9 months following diagnosis (53, 54). Approximately 4% to 7% of patients with colorectal cancer present with peritoneal carcinomatosis despite early detection, with the peritoneum serving in second place to the liver as a site of metastasis (55). MCT4 was strongly expressed in cells from malignant ascites of colorectal cancer in our study; thus, this result indicates that an MCT4 inhibitor may overcome treatment failure in patients with peritoneal carcinomatosis. However, we observed that the expression of basigin could be decreased by MCT1 and MCT4 inhibition. The possible role of basigin in peritoneal carcinomatosis could not be excluded.

In conclusion, the result of this study showed that MCT4 was expressed in colorectal cancer and most notably in PDCs from malignant ascites, and that it may serve as a factor indicating poor prognosis in this disease. MCT4 inhibitor might be a key treatment for hypoxic tumors in conjunction with radiotherapy or chemotherapy. Further studies, such as clinical trials to elucidate the benefit of MCT4 inhibitor treatment and the role of basigin, are therefore warranted.

Acknowledgments

This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number; HI14C3418).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

Safety and efficacy of oxaliplatin and fluoropyrimidine regimens with or without bevacizumab as first-line treatment of metastatic colorectal cancer: results of the TREE Study.
J Clin Oncol2008;26:3523–9.